Loss of the collagen IV modifier prolyl 3-hydroxylase 2 causes thin basement membrane nephropathy

The glomerular filtration barrier (GFB) produces primary urine and is composed of a fenestrated endothelium, a glomerular basement membrane (GBM), podocytes, and a slit diaphragm. Impairment of the GFB leads to albuminuria and microhematuria. The GBM is generated via secreted proteins from both endothelial cells and podocytes and is supposed to majorly contribute to filtration selectivity. While genetic mutations or variations of GBM components have been recently proposed to be a common cause of glomerular diseases, pathways modifying and stabilizing the GBM remain incompletely understood. Here, we identified prolyl 3-hydroxylase 2 (P3H2) as a regulator of the GBM in an a cohort of patients with albuminuria. P3H2 hydroxylates the 3′ of prolines in collagen IV subchains in the endoplasmic reticulum. Characterization of a P3h2ΔPod mouse line revealed that the absence of P3H2 protein in podocytes induced a thin basement membrane nephropathy (TBMN) phenotype with a thinner GBM than that in WT mice and the development of microhematuria and microalbuminuria over time. Mechanistically, differential quantitative proteomics of the GBM identified a significant decrease in the abundance of collagen IV subchains and their interaction partners in P3h2ΔPod mice. To our knowledge, P3H2 protein is the first identified GBM modifier, and loss or mutation of P3H2 causes TBMN and focal segmental glomerulosclerosis in mice and humans.


Urine collection and urinalysis 83
To collect mouse urine, animals were held over a Petri dish and slight pressure was 84 exerted manually on the bladder. The urine collected in the Petri dish was picked up with 85 a pipette and transferred into a 1.5 ml Eppendorf tube. The urine was then stored at -20 86 °C until further evaluation or analyzed freshly after centrifugation to evaluate for red blood 87

cells. 88
A homemade albumin ELISA was used for albumin measurement in the urine of mice at 89 6w, 28w and 48w. In short, a 96 well plate was coated with goat anti-mouse albumin 90 antibody (Bethyl, Cat# A90-134A) in coating buffer (0.05M Carbonate-bicarbonate pH 91 9.6) as 1:100 dilution overnight at 4 °C. The following day, the coated plate was incubated 92 with a post coat solution (50mM TBS, 1%BSA pH 8.0) for 30 min. Afterward, the plate 93 was incubated with diluted urine (1:300) for 1h. After washing, the plate was incubated 94 with the secondary antibody, goat anti-mouse Albumin-HRP, (Bethyl, Cat# A90-134P) for 95 1h. TMB substrate (Biomol, Cat# E102) was incubated for 3 min and the reaction was 96 stopped by stop solution (2N H2SO2). The absorbance was measured at 450 nm in an 97 ELx808 Absorbance Microplate Reader from Biotek Instruments. 98 A creatinine measurement kit (Labor und Technik, Cat# LT-CR 0106) was used according 99 to the manufacturer's protocol to measure creatinine in mice urine at 6w, 28w and 48w. 100 The measurement was taken at 562 nm in a TECAN Sunrise Basic microplate reader. the manufacturer's protocol to measure urea in mice serum at 28w and 48w. The 103 measurement was taken at 365 nm in a Thermo Fisher NanoDrop spectrophotometer. 104 A cystatin C measurement kit (Abcam, Cat# ab201280) was used according to the 105 manufacturer's protocol to measure cystatin C in mice serum at 28w and 48w. The 106 measurement was taken at 450 nm in a TECAN Sunrise Basic microplate reader. 107 Mouse urine at 48w was evaluated under a light microscope to count red blood cells at 108 40x magnification for hematuria. Dipsticks were used for qualitative measurement 109 (Siemens Multistix 10SG, Cat# 01526748). 110

Immunohistochemistry 181
After deparaffinization and rehydration of tissue sections, the slides were immersed in 1% 182 periodic acid solution for 15 min at RT and stained with Schiff's reagent for 45 min at RT. 183 For counterstain, tissues were stained with haematin for 3 min at RT. The slides were 184 immersed in 50% ethanol, 70% ethanol, 95% ethanol, 100% ethanol, and covered with 185 Eukitt to fix the staining. Images were taken at the Zeiss Axio Scope A1 microscope. were then incubated in a humidified oven at 37°C for 2h to complete gelation. Next, the 224 gelling chambers were removed and the tissue sections were incubated in 8U/ml 225 proteinase K (Sigma-Aldrich, Cat# P2308-100MG) in a Tris/EDTA-based digestion buffer 226 (50mM Tris (pH 8), 25 mM EDTA, 0.5% Triton X-100 and 0.8 M NaCl) at 60°C for 4h. The 227 digested tissue sections were then removed from the slide and placed in doubly deionized 228 water at room temperature for 60 min for isotropic expansion. After expansion, the tissue 229 sections were removed from the doubly deionized water and mounted in glass-bottom 230 chamber slides (Ibidi µ-Slide 2 Well Glass Bottom) for subsequent super-resolution 231 imaging. 232

Imaging 233
Post-expansion super-resolution imaging was performed using a Zeiss LSM 800 confocal 234 microscope with Airyscan using the optimized 63x objective and 8x digital zoom with 235 subsequent Airyscan processing. Fiji imaging software (Max Planck Institute of Molecular 236 Cell Biology and Genetics) was used to navigate the files and to adjust color balance. 237 238 Transmission Electron microscopy 239 TEM analysis was applied as previously described (4). In summary, tissues were fixed 240 with 4% PFA and 1% glutaraldehyde and 1-2 mm 3 blocks of the kidney were cut. After 241 dehydration, the tissues were embedded in epoxy resin (Durcupan ACM,Fluka, Aldrich, Gillingham, UK). 40 nm ultrathin sections were cut and analyzed using a Phillips 243 CM 100 transmission electron microscope. For quantitative analysis, 4 mice of each time 244 point and genotype were analyzed. 15 to 20 random images of glomeruli were taken at a 245 magnification of 2900x using a Phillips CM 100 transmission electron microscope. The 246 Image J software were used to create an overlay using gridlines with a mesh size of 247 500nm randomly placed on top of the randomly taken images. Then, GBM thickness at 248 each crossing point of the grid lines were measured with the GBM. For Bowman capsule 249 we did the same however here we measured only 4 to 7 randomly taken images per 250 animal and the mesh size was 1µm. Determination of foot process width was performed 251 as previously described and image analysis was done via ITEM software (Olympus) or 252 ImageJ software (5). 253

Construction and production of AAV vectors 254
The human P3H2 gene (accession number NM_018192.4) was amplified by PCR from a 255 plasmid encoding P3H2 cDNA (GenScript, Cat# OHu02725) with the forward primer AgeI: Migration assay 283 20.000 cells per well were split into a culture-Insert 2 Well in µ-Dish 35 mm (Ibidi, Cat# 284 81176) and images were taken at 0h, 3h, 6h, and 9h. The cell-free area was measured 285 by the ImageJ program to calculate the migrated area. 286 Extracellular matrix isolation 287 12 days differentiated P3H2 KO and WT immortalized human podocyte cell lines were 288 lysed with alkaline detergent buffer (20mM NH4OH and 0.5% v/v TritonX-100 in PBS) for 289 1 min at 37° C and washed with 1x PBS. After incubation with 10 µg/ml DNase I (Roche, 290 Cat# 10104159001) for 1h at 37 °C, denuded ECM was scraped into reducing buffer 291

Statistical data analysis 359
The data in the diagrams of the results section are shown in different types of plots. All 360 statistical analysis were performed and plots were prepared by using GraphPad Prism 361 (v8.4.0). The data are presented as mean with SD or median with IQR. Two-tailed 362 Student's t-test and Mann Whitney's U test were used to test for significance between 363 experimental and control group. When three or more groups were assessed, one-way 364 ANOVA with Tukey multiple comparison post hoc tests was used. A p-value of p <0.05 365 (*) was set as the significance level. A p-value of p <0.01 (**) was found as very significant 366 and a p-value of p <0.001 (***) and p <0.0001 (****) were found as highly significant. 367 368